U.S. patent application number 12/270621 was filed with the patent office on 2009-07-09 for apparatus and method for the purification of biomolecules.
This patent application is currently assigned to STRATEC BIOMEDICAL SYSTEMS AG. Invention is credited to Peter Bendzko, Ralf Griebel, Hans Joos.
Application Number | 20090176308 12/270621 |
Document ID | / |
Family ID | 40844902 |
Filed Date | 2009-07-09 |
United States Patent
Application |
20090176308 |
Kind Code |
A1 |
Griebel; Ralf ; et
al. |
July 9, 2009 |
APPARATUS AND METHOD FOR THE PURIFICATION OF BIOMOLECULES
Abstract
The invention comprises an apparatus 10 and a method 100 for
automatically lysing, extracting and purifying biomolecules 28. As
the central technology for purifying the biomolecules magnetizable
particles 35 and an adapted magnet device 40 are used for binding
and transferring the particles 35. The apparatus 10 comprises a
highly efficient incubation unit 60 with options for upscaling the
maximum sample amount. Furthermore the apparatus 10 in particular
comprises a group of magnetizable pins 40 as transport magnets and
furthermore counter magnets 50 which are arranged on the cavities
20, in which solutions 30 with the biomolecules 28 to be lysed and
magnetizable particles 35 are disposed. The use of counter magnets
50 improves the quality of the eluate. Furthermore the invention
comprises a fully automatic system 800 and a method 100 for
controlling the process steps and the selection of the reagents and
the aids (e.g. magnetizable particles 35) on the basis of
automatically detected information, such as gathered e.g. from the
loaded samples and/or from the used reagents. The automated overall
system 800 in particular fulfils the requirements of medical
diagnostics.
Inventors: |
Griebel; Ralf; (Birkenfeld,
DE) ; Joos; Hans; (Berlin, DE) ; Bendzko;
Peter; (Berlin, DE) |
Correspondence
Address: |
INTELLECTUAL PROPERTY / TECHNOLOGY LAW
PO BOX 14329
RESEARCH TRIANGLE PARK
NC
27709
US
|
Assignee: |
STRATEC BIOMEDICAL SYSTEMS
AG
Birkenfeld
DE
|
Family ID: |
40844902 |
Appl. No.: |
12/270621 |
Filed: |
November 13, 2008 |
Current U.S.
Class: |
436/8 ;
422/400 |
Current CPC
Class: |
B01F 11/0082 20130101;
C12N 15/1013 20130101; Y10T 436/10 20150115; G01N 35/0098
20130101 |
Class at
Publication: |
436/8 ; 422/100;
422/101 |
International
Class: |
G01N 1/34 20060101
G01N001/34; B01L 3/02 20060101 B01L003/02; B01L 11/00 20060101
B01L011/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 13, 2007 |
DE |
10 2007 054 033.9 |
Claims
1.-34. (canceled)
35. A system for diagnostically purifying biomolecules comprising:
a plurality of cavities for accommodating solutions with
magnetizable particles, at least one magnetizable pin arranged in
such a fashion that it can be inserted in at least one of the
cavities, a control unit for controlling the system, wherein the
system yields an eluate with diagnostically purified biomolecules
from the sample material, and a detection unit for recognizing a
coding and for transferring the coding to a yield cavity after the
diagnostically purifying.
36. The system according to claim 35 furthermore comprising: a
loading bay for accommodating substances, wherein the substances
are at least one of a sample material, a buffer solution or
magnetizable particles, a reading module for automatically
identifying the substances and the sample material upon
accommodating in the loading bay, an incubation unit for heating,
thermostating and incubating the samples in a lysis cavity, a
pipetting unit for transferring the substances, a receptacle, and
at least one yield cavity.
37. The system according to claim 35, furthermore comprising:
apparatus for improving the quality of the eluate before removing
by pipetting adapted to keep the magnetizable particles away from a
tip of the pipetting device.
38. The system according to claim 35, furthermore comprising:
sensors for recording and logging a plurality of parameters using a
documentation unit.
39. The system according to claim 35, furthermore comprising: a
plurality of parameter controls for controlling the plurality of
parameters using the control unit.
40. The system according to 36, wherein the control unit is adapted
to check a consistency of the substances disposed in the loading
bay based on the substances in the loading bay.
41. The system according to claim 40, wherein the control unit is
adapted to check, if the consistency of the substances disposed in
the loading bay is correct based on a composition of a kit.
42. The system according to claim 40, wherein the control unit is
adapted to output a message in the case that the consistency of the
substances in the loading bay is not correct.
43. The system according to claim 40, wherein the control unit is
adapted to prevent an opening of the loading bay during the
controlling of the system, in case that the consistency of the
substances disposed in the loading bay is correct.
44. The system according to claim 36, wherein the system is adapted
to determine a substance amount of required buffer solutions in the
loading bay based on the identified substance in the loading
bay.
45. The system according to claim 36, wherein the system is adapted
to determine a substance amount of the required magnetizable
particles based on the identified substance in the loading bay.
46. The system according to claim 36, wherein the system is adapted
such that the system determines a number and a sequence of process
steps of a method for diagnostically purifying biomolecules based
on the identified substance in the loading bay.
47. The system according to claim 36, wherein the system is adapted
to determine an appropriate incubation volume for incubating based
on the identified substance in the loading bay.
48. The system according to claim 47, wherein the system is adapted
to determine a suitable multiwell plate for incubating based on the
appropriate incubation volume.
49. The system according to claim 35, wherein the system is adapted
to automatically select, request and use a suitable cavity from the
group consisting of lysis cavity, incubator cavity, work plate,
cavity of the work plate, yield plate.
50. A method for diagnostically purifying biomolecules comprising:
mixing the biomolecules with a binding buffer and magnetizable
particles thus forming a particle-biomolecule complex, wherein the
biomolecules are bound by the binding buffer to the magnetizable
particles, switching on at least one magnetizable pin, transporting
the particle-biomolecule complex to a first cavity with a first
solution, switching off the magnetizable pin, mixing the
particle-biomolecule complex with the first solution; for yielding
an eluate with the diagnostically purified biomolecules from a
sample material subjected to the effect of magnetizable
particles.
51. The method according to claim 50, wherein the method
furthermore comprises: inserting at least one sample cavity with
the sample material in the loading bay, identifying the sample
material upon accommodation in the loading bay, inserting at least
one substance cavity with a substance in the loading bay,
identifying at least one substance upon accommodation in the
loading bay, checking a consistency of the substances in the
loading bay with the sample material, transferring the substances
and/or the sample material.
52. The method according to claim 50, the method furthermore
comprising: recording a plurality of parameters, controlling the
plurality of parameters.
53. The method according to claim 50, furthermore comprising:
determining a plurality of process parameters based on the at least
one identified substance.
54. The method according to claim 53, wherein the determining of
the plurality of process parameters based on the at least one
identified substance comprises: determining at least one buffer
solution, determining one required magnetizable particle,
determining one required elution solution.
55. The method according to claim 50, furthermore comprising
determining one substance amount of the substances, determining
required parameter values for executing the method, determining a
number and a sequence of process steps of the method for
diagnostically purifying biomolecules, determining a suitable
incubation volume, determining a suitable multiwell plate for
incubating based on the suitable incubation volume.
56. The method according to claim 55, wherein determining the
substance amount of the substances comprises: determining one
substance amount of at least one buffer solution, determining one
substance amount of one magnetizable particle, determining one
suitable elution solution.
Description
[0001] This application claims the priority from the German patent
application DE 10 2007 054 033.9, to which reference is made and
which in its entirety forms part of this disclosure. This
application is filed concurrently in Germany and the USA.
FIELD OF THE INVENTION
[0002] The invention relates to a modular apparatus and a composite
multistage method as well as a comprehensive system for the
automated lysis, extraction and purifying of biomolecules (e.g.
DNA, RNA or proteins). The method steps according to the invention
comprise recognizing substances, placing the substances in
cavities, lysis, binding or adsorbing of biomolecules to
magnetizable particles, transferring particles, washing and
cleaning of biomolecules, eluting or desorbing the biomolecules,
removing the magnetizable particles and pipetting the eluate into a
yield cavity. The system fulfils the requirements of clinical
in-vitro diagnostics.
DESCRIPTION OF RELATED ART AND BACKGROUND OF THE INVENTION
[0003] There are a very large number of suppliers of apparatus and
methods for the purifying of biomolecules via adsorption to
magnetizable particles. The known methods are determined by the
type of biomolecules and their interaction with the magnetizable
particles. These types of purifying methods are in particular
adapted for the automation of the methods for purifying of
biomolecules.
[0004] Among the suppliers of apparatus and methods for the
purifying of biomolecules the following suppliers are known:
Chemagen, Qiagen, Thermo, Promega, Roche Diagnostics, Agowa, Dynal,
Thermo Fisher, Analytik Jena and Tecan.
[0005] These companies partly protected their apparatus through
their own patents (for example WO 03/044537, WO 2007/020294, U.S.
Pat. No. 6,448,092 and WO9609550).
[0006] Most of these known apparatus have in common that they are
so-called magnet separators without pipetting function (Thermo
Kingfisher, Promega, Agowa). This means an operator pipettes
solutions prior to the extraction process by hand or he uses
pre-filled plastic cavities for the extraction. In an apparatus
supplied by Chemagen the cavities are also filled, but the eluate
still needs to be removed by pipetting manually. These approaches
are sufficient for applications in a field of research, however,
they are not sufficient in the diagnostic field.
[0007] An apparatus offering a further solution is the Magnapure
technology by Roche Diagnostics.
[0008] The European patent application No. 0 644 425 by Hoffmann-La
Roche discloses an analysis apparatus with a device for separating
magnetic microparticles from a suspension. The apparatus disclosed
in the Hoffmann-La Roche application comprises two permanent
magnets between which a reaction cavity containing a suspension is
disposed. The permanent magnets are arranged diametrically opposite
each other with respect to the reaction cavity. The polar axes of
the permanent magnets and the longitudinal axis of the reaction
cavity form an acute angle. With this arrangement of the polar axes
the magnetic stray field can be used for the separation of the
magnetic microparticles, thereby accelerating the separation of the
magnetic microparticles.
[0009] U.S. Pat. No. 6,596,162 (Thermo Labsystems Oy) and U.S. Pat.
No. 6,040,192 (Labsystems Oy) disclose an apparatus for separating
magnetic particles from a reaction cavity with a removable
permanent magnet.
[0010] The use of permanent magnets and suspensions with
magnetizable particles is the central technology of the known
apparatus. Due to the effect of the magnetic force the magnetizable
particles are collected from the suspension and adhere to the
magnet. After removing the magnetic field these particles are
released again. This object is solved in different ways in the
known apparatus.
[0011] U.S. Pat. No. 6,409,925 (Bio-Magnetics Ltd.) discloses an
apparatus for the collecting of magnetic particles as well as the
transfer of the magnetic particles from a first cavity to a second
cavity. For collecting and transferring the magnetic particles this
disclosure suggests the use of a mobile magnetic element in order
to magnetize a tip. A magnetization of the tip occurs upon
approaching the mobile magnetic element to the tip. The magnetized
tip is adapted for collecting and the transferring the magnetic
particles.
[0012] EP 1 726 963 A2 (Festo Corporation) discloses a system for
transferring a sample material from a source cavity to a target
cavity. A transfer unit comprises at least one pin tip with a
central bore and a magnetic actuator element being moveable between
a first and a second actuator position. The sample material in
proximity of the pin tip is either collected or released by moving
the actuator element. Furthermore a group of counter magnets is
provided underneath the source cavity and/or the target cavity, in
order to support the transfer of the sample material. The Festo
system furthermore allows a common movement of the actuator element
and the group of counter magnets in order to mix the sample
material. It is furthermore provided that each pin tip is
individually controllable; likewise, the movement of each of the
counter magnets is individually controllable.
[0013] The above-mentioned solutions have the disadvantage of
comprising qualitative shortcomings in the production of the
eluate; and that their majority does not fulfill the requirements
of in-vitro diagnostics, comprehensively.
[0014] The purifying of biomolecules using the magnetizable
particles leads to "residual particles" in the eluate according to
the methods known in the state of the art. These have to be removed
at a great effort in a further process step, for example by
centrifugation, since they impair subsequent analytic
processes.
[0015] Furthermore, the concatenation of the process steps from the
lysis of the sample to the selection of the substances and aids,
via the numerous processing steps yielding the final eluate so far
comprised numerous sources of errors typically leading to
contamination, falsely negative or falsely positive results.
[0016] Furthermore, there is the danger of confusion when combining
substances within a kit used for the so-called purifying. A kit
consists of a defined combination of substances and aids. The
substances and aids comprise e.g. elution buffers. A use of e.g.
wrong elution buffers would lead to the biomolecules no longer
being separable from the biomolecules bound to the magnetizable
particles in the elution buffer, and would thus erroneously no
longer be detectable in the eluate. It is essential to prevent
this.
[0017] The present invention uses among others kits of substances
for the purifying of nucleic acids which are available for example
from Invitek (disclosed e.g. in the patents: WO01/70386,
WO2004/042058, DE10253351, WO00/34463, WO99/32616, U.S. Pat. No.
6,037,465, DE59610721, DE4422044 and others). Specifically, the
invention uses magnetizable particles adapted to temporarily bind
the biomolecules, thus rendering them transportable.
[0018] These kits of substances by Invitek require, in particular
during the lysis, temperatures of up to 90.degree. Celsius over a
period of for e.g. 20 minutes, wherein sometimes large sample
volumes have to be processed. These requirements are not fulfilled
by conventional incubation systems. Rather, special aspects of
incubators are required.
[0019] Furthermore the required sample volumes vary very strongly
to yield a desired amount of biomolecules depending on the field of
application. Consequently, in practice problems have frequently
occurred with apparatus that can only cover a predetermined, narrow
volume range in automated processing.
SUMMARY OF THE INVENTION
[0020] The apparatus for a purifying of biomolecules comprises a
plurality of cavities for accommodating solutions with magnetizable
particles. Furthermore the apparatus comprises at least one
magnetizable pin that is arranged such that it is insertable in at
least one of the cavities, and at least one counter magnet is
arranged in at least one bottom area of the cavities.
[0021] The lysis cavities 22 and the work plate 80 may be used as
cavities within the apparatus 10. Optionally also the yield plate
85 may serve as cavities being arranged outside the apparatus 10
forming a component of the system.
[0022] The apparatus and the method according to the invention
serve for the completely automated lysis, extraction and purifying
of biomolecules (e.g. DNA, RNA or proteins) up to the final eluate
containing the biomolecules in an ultrapure suspension, thereby
also corresponding to the requirements of clinical in-vitro
diagnostics (IVD). The samples are of a highly different nature.
The application of the invention reaches from the field of research
to the field of IVD. The purifying occurs by adsorption of the
biomolecules to magnetizable particles. A complete run of the
method according to the present invention is rendered possible
within the apparatus. The apparatus executes the complete method
for the lysis, extraction and purifying for the corresponding
biomolecules without any intervention by laboratory staff. This
means that at the start the sample (e.g. blood, patient tissue,
blood plasma, blood serum), bearing a machine-readable
identification, is placed into the apparatus. Selectively, the
sample is attributed to an unambiguously identified processing job.
Selectively on the basis of this machine-readable job or based on
an input program the apparatus controls the addition of the
substances and aids and derives all required process parameters for
controlling the method the identification of the sample, the
substances and the aids. The complete process is documented and
takes place without possibility of interference by the laboratory
staff. This means that there is no danger of confusion, a strongly
reduced danger of infection, no danger of contamination and
cross-contamination.
[0023] The present invention is adapted to use a plurality of
cavities for accommodating mixtures of samples and reagents. The
invention discloses a method for enhancing the minimum and maximum
sample volumes and also the amount of processable biomolecules
(upscaling).
[0024] Moreover, the invention discloses a closable incubation unit
adapted to generate a homogeneous temperature within the
cavities/in the mixture of samples and reagents in the incubator
within a few minutes, and is adapted to stabilize this temperature
with great precision without any liquid evaporating.
[0025] The invention furthermore discloses a method for lysing and
purifying biomolecules within this mixture of samples and reagents
using magnet particles or magnetizable particles. This method
comprises a mixing of the biomolecules with a binding buffer and
magnetizable particles, thereby forming a particle-biomolecule
complex, wherein the biomolecules are bound to the magnetizable
particles by the binding buffer.
[0026] The method furthermore comprises the switching on or
inserting of a magnetizable pin. Moreover the method comprises the
transporting of the particle-biomolecule complex to a first cavity
with a first solution. In addition the method comprises the
switching off of the magnetizable pin, an optional switching on of
a counter magnet and a mixing of the particle-biomolecule complex
with the first solution.
[0027] The inventive method and the apparatus for purifying, use
two groups of magnets for the purifying of biomolecules after their
adsorption to magnetizable particles: a plurality of magnetizable
pins and at least one counter magnet. Thereby it is in particular
possible to substantially reduce the number of magnetizable
"residual particles" present in the eluate; the group of counter
magnets allows the retention of the "residual particles" present in
the eluate in the cavity. Thereby the purity of the eluate and
consequently the functionality of subsequent analyses are
improved.
[0028] The invention furthermore discloses a system for lysis,
extraction and diagnostically purifying of biomolecules. The system
according to the invention comprises the apparatus for purifying of
biomolecules according to the invention. The system furthermore
comprises a control unit for controlling the apparatus. The system
yields an eluate with diagnostically purified biomolecules from the
sample material. Moreover, a detection unit detects a for detecting
a code: The detection unit furthermore transfers the code to a
yield cavity.
[0029] Furthermore the invention discloses a method for the
diagnostically purifying of biomolecules comprising an execution of
the method for purifying biomolecules according to the invention to
yield an eluate with the diagnostically purified biomolecules from
a sample material under the exposure to magnetizable particles.
FIGURES
[0030] FIG. 1a shows a perspective view from above of the apparatus
according to the invention
[0031] FIG. 1b shows a perspective view from below of the apparatus
according to the invention
[0032] FIG. 1c shows a cross section of the apparatus according to
the invention
[0033] FIG. 2 shows the flow of the method according to the
invention as a flow chart
[0034] FIG. 3a shows an example of a plate with large incubator
cavities in 12 rows from above
[0035] FIG. 3b shows an example of a plate with 8 times 12 equaling
96 incubator cavities from above
[0036] FIG. 3c shows a method without upscaling
[0037] FIG. 3d shows a method with an upscaling from a large
cavity
[0038] FIG. 3e shows a method with an upscaling from standard
cavities
[0039] FIG. 3f shows a method with an upscaling from standard
cavities
[0040] FIG. 4a shows a perspective lateral view of an alternative
aspect of the apparatus for lysis and extraction
[0041] FIG. 4b shows a perspective view from behind of the
alternative aspect of the apparatus for lysis and extraction
[0042] FIG. 4c shows a cross section of the alternative aspect of
the apparatus
[0043] FIG. 4d shows a magnetizable pin according to the
invention
[0044] FIG. 5a shows a top view of a system according to the
invention
[0045] FIG. 5b shows a perspective view of the system according to
the invention
[0046] FIG. 6a shows a method for diagnostically purifying
biomolecules
[0047] FIG. 6b shows a section of the method for diagnostically
purifying biomolecules
[0048] FIG. 7 shows a block diagram of the system according to the
invention
DETAILED DESCRIPTION OF THE INVENTION
[0049] The invention will now described on the basis of the
drawings. It will be understood that the embodiments and aspects of
the invention described herein are only examples and do not limit
the protective scope of the claims in any way. The invention is
defined by the claims and their equivalents. It will be also
understood that features of one aspect can be combined with
features of a different aspect.
[0050] FIG. 1a-c show a perspective view of the apparatus 10
according to one aspect of the invention. The apparatus 10 on one
level comprises a row of fasteners or a rack 44 for disposables,
e.g. plastic covers 42, an incubation unit 60, a work-area retainer
81 for a work plate 80, an accommodation cavity 70 for the used
disposables, as well as a yield-area retainer 86 for a yield cavity
85. A first drive motor 90a and a first band 92a are mounted on the
level. The work-area retainer 81 and the yield-area retainer 86
each have a plurality of cavities 20.
[0051] The apparatus 10 has a frame 45 with magnetizable pins 40
with electric connections 46. The frame 45 comprises a second drive
motor 90b, a second band 92b and a spindle 92c.
[0052] Below the level, below the work-area retainer 81 counter
magnets 50 with electric connections 51 are disposed (see FIG. 1b).
Racks 44 for accommodating a plurality of the disposables 42 are
provided next to the incubation unit 60.
[0053] The disposables used are preferably plastic covers 42 and
consist of for e.g. polypropylene or any other suitable material.
For example the aspect of the plastic covers 42 described within
the framework of this description is approx. 50.5 mm long and has a
diameter of approximately 5.5 mm, widening to approx. 8.5 mm toward
the upper end. The wall thickness is approximately 0.5 mm. Of
course also different dimensions of the plastic covers 42 are
possible. The dimensions of the plastic covers 42 are chosen such
that they fit ends 41 of the magnetizable pins 40 well.
[0054] Of course also other dimensions of the plastic covers 42 are
possible, as long as they fit the ends 41 of the magnetizable pins
40 well and can still dip into the cavities 20 comfortably, without
making liquids in the cavities 20 flow over.
[0055] For example the magnetizable pins 40 have a bulge 43 (see
FIG. 1c) on which the plastic covers 42 are caught. The plastic
covers 42 are disposed in a rack 44 for plastic covers 42 adapted
to accommodate plastic covers 42 and is optionally filled by an
automatic feeding system (not shown in FIGS. 1a, 1b and 1c).
[0056] The incubation unit 60 can accommodate at least one lysis
cavity 22 (not shown) in a sample area. The start material or
sample material containing biomolecules 28 is placed in the lysis
cavity 22. In a fully automated or semi-automated process the
filling of the lysis cavity 22 preferably takes place using a
suitable pipetting system, which is not shown in FIGS. 1a and 1b,
as will be explained below in connection with the system according
to the present invention (see FIG. 7a).
[0057] Optionally the incubation unit 60 can be closed by a lid 61,
as visible in FIG. 4a, or by a lid 61 with openings 62, as visible
in FIG. 1a. Into these openings 62 in the lid 61 of the incubation
unit 60 a suitable pipetting system 840 can be lowered (see FIG.
5b). The incubation unit 60 allows a heating of the sample area to
temperatures above the room temperature. Preferably the openings 62
of the incubation unit 60 are designed such that the openings 62
can be closed by lowering the magnetizable pins 40, which
facilitates achieving a target temperature within the lysis cavity
22 inside of the incubation unit 60. Additionally moving the
magnetizable pins 40 up and down in the lysis cavity 22 effects a
mechanical mixing of the start material of biomolecules 28 and at
least one lysis buffer 30a and added magnetizable particles 35, in
order to form a solution 30. The lysis buffer 30a and the
magnetizable particles 35 were filled into the lysis cavity 22
beforehand by the suitable pipetting system 840 (shown on the right
in FIG. 5b).
[0058] It is furthermore possible to optionally automatically
remove the optional lid of the incubation unit 60 after a
selectable time interval, to facilitate the cooling of the solution
30. Additionally or alternatively the cooling process can also be
supported by heat-dissipating procedures, e.g. a ventilator.
[0059] The work plate 80 can be implemented as a deep-well plate
and can contain a first plurality of the cavities 20. For a
conventional deep-well plate, each cavity 20 can hold a volume of
approx. 2 ml, which is a suitable size to accommodate for example
blood samples and to purify biomolecules 28, e.g. DNA or RNA
material. This volume allows integrating this apparatus 10 in a
fully automated process for purifying biomolecules 28.
[0060] The yield cavity 85 can be implemented as a microtiter plate
for accommodating purified biomolecules 28 in the solutions 30.
Alternatively the yield cavity 85 can also be implemented by one or
several lines of individual sample tubes accommodating the purified
biomolecules 28.
[0061] A rake can be provided to facilitate the slipping off of the
disposables above the accommodation cavity 70, for used
disposables; in particular the plastic covers 42.
[0062] According to a further aspect of the present invention it
may be of interest to arrange the accommodation cavity 70 for used
disposables between the work plate 80 and the yield cavity 85. It
is thus ensured that the disposables used in the purification
process do not have to be moved above the yield cavity 85, thereby
further reducing unintentional contamination of the purified
biomolecules 28.
[0063] The frame 45 is moved along one side of the apparatus 10, so
that the frame 45 can sweep the rack 44 for plastic covers 42, the
incubation unit 60, the work plate 80, the yield cavity 85, the
accommodation cavity 70 for used plastic covers 42. In the aspect
of the apparatus 10 shown in FIG. 1a-c the frame 45 is moved along
the longitudinal side of the apparatus 10.
[0064] The frame 45 is moved by the first actuator 90a. Preferably
the frame 45 is implemented as a sliding carriage on two
longitudinal rails 92 and the thrust of the first drive motor 90a
is transferred to the carriage slide via the first band 92a, so
that the frame 45 can be moved along the longitudinal axis of the
apparatus 10. The first drive motor 90a can be controlled by
suitable software.
[0065] The magnetizable pins 40 can be implemented as permanent
magnets. Alternatively it is possible to implement the magnetizable
pins 40 as electromagnets. The necessary electric connections 46
for controlling the magnetizable pins 40 implemented as
electromagnets are shown in FIG. 1a.
[0066] By means of the second drive motor 90b the magnetizable pins
40 can be moved on the frame 45 in the z direction. In the
embodiment of the apparatus 10 shown in FIG. 1a-c the movement of
the second drive motor 90b is transferred to the spindle 92c via
the second band 92b, so that the z position of the magnetizable
pins 40 can be varied. The movement of the second drive motor 90b
can also be controlled by software.
[0067] A movement of the magnetizable pins 40 along the z direction
can be used for mixing the solution 30 within the cavity 20. For a
magnetizable pin 40 implemented as a permanent magnet dipping the
magnetizable pin 40 into the solution 30 within the cavity 20
generates a magnetic field to which the solution 30 is subjected.
In case that the magnetizable pin 40 is implemented as an
electromagnet, in addition to lowering the magnetizable pin 40 a
current flow of suitable amplitude and direction has to be
circuit-switched through the electromagnet, in order to switch the
magnetizable pin 40 to magnetic or non-magnetic. Also in this case
the moving up and down of the magnetizable pin 40 in the z
direction serves for mixing the solution 30 or to improve the
collection of magnet particles 35 within the cavity 20.
[0068] In the alternative aspect with permanent magnets instead of
electromagnets a removal of the magnetizable pin 40 from the
solution, corresponds to a switching off of the magnet or of the
magnetic field of the magnetizable pins 40. Outside the solution 30
the magnetizable pin 40 does not exert a magnetic field on the
solution 30 within the cavity 20.
[0069] In the aspect of FIG. 1a-c the counter magnets 50 are
arranged below the work plate 80, near the bottom area 25 of the
cavities 20 within the work plate 80. The counter magnets can be
identified best in FIGS. 1b and 1c. The group of counter magnets 50
comprises at least one counter magnet 50. The counter magnets 50
can be implemented as an arrangement of permanent magnets, so that
respectively one of the counter magnets 50 corresponds to one
cavity 20 within the work plate 80. The position of the counter
magnets 50 can be changed relative to the cavities 20 of the work
plate 80. In such an aspect it is possible to approach the cavities
20 with the permanent magnets, so that the cavity 20 is penetrated
by the magnetic field of the counter magnets 50 in the bottom area
25.
[0070] In a second position of the permanent magnets the counter
magnets 50 are sufficiently spaced apart from the cavities 20, so
that the magnetic field of the counter magnets 50 essentially
vanishes in the bottom area 25 of the cavities 20. The magnetic
field of the counter magnets 50 thus can be switched by a suitable
position of the counter magnets 50 implemented as permanent
magnets. As for the aspect according to FIG. 1b counter magnets 50
are allocated to the cavities 20. In FIG. 5b one of the counter
magnets 50 is allocated to several of the cavities 20. Likewise it
would be possible to allocate to each of the cavities 20 an
individual one of the counter magnets 50.
[0071] Alternatively it is in addition possible to provide only one
row of a plurality of counter magnets 50, so that the number of
counter magnets 50 corresponds to the number of columns within the
work plate 80. For such an aspect the apparatus 10 comprises a
device for moving the row of counter magnets 50 with the movement
of the frame 45 and thereby the row of magnetizable pins 40. It
would be possible to couple the movement of the row of counter
magnets 50 to the movement of the first band 92a. In the case that
the row of counter magnets 50 is embodied as permanent magnets, it
is again necessary that the row of counter magnets 50 is mobile in
order to switch the magnetic field in the bottom area 25 of the
cavities 20 of the work plate 80 either on or off.
[0072] Moreover it is possible to implement the counter magnets 50
only as one single counter magnet 50 which is moved from one cavity
20 to the next and moves along a grid in the x and y directions to
cover all the cavities 20. However, for a system with a plurality
of magnetizable pins 40 such a method is slower than the two
above-mentioned aspects of the counter magnets 50.
[0073] Independent of the special aspect of the counter magnets 50
these can be implemented as electromagnets which, upon a current
flow, induce a magnetic field in the bottom area 25 of the cavities
20. This magnetic field of the counter magnets 50 vanishes
substantially as soon as no current flows through the
electromagnets any longer. Hysteresis effects within the
electromagnets as well as diffusion processes of the magnetizable
particles 35 within the solutions 30 limit the speed at which the
counter magnets 50 implemented as electromagnets can be switched.
The polarity of the current flow is to be chosen in such a fashion
that, when the magnetic field is active, the magnetizable particles
35 are subjected to a force in the direction of the bottom area 25
of the cavity 20. For an aspect of the apparatus 10 containing
electromagnets as counter magnets 50 it is consequently possible to
switch the magnetic field of the counter magnets 50 on and off
depending on the current flow. It is of interest to have the
software controlling the apparatus 10 to switch the counter magnets
50 on and off. Counter magnets 50 implemented as electromagnets are
shown in FIG. 1b with their electric connections 50a.
[0074] In FIG. 1c furthermore the solutions 30 within the cavities
20 are shown. The solutions 30 can still contain residual amounts
of magnetizable particles 35 even after the last stage of the
eluate production. The movement of these magnetizable particles 35
toward the bottom area 25 of the cavities 20 is supported by the
counter magnets 50. The effect of the counter magnets generates a
shift of the residual magnetizable particles 35 toward the bottom
and thereby improves the eluate to be free from particles, which is
transferred from the upper layer out of the cavity 20 into the
eluate cavity 85 using the pipetting device 840.
[0075] FIG. 2 shows a flow of a method 100 according to the
invention for purifying biomolecules 28. The apparatus 10 and the
method 100 according to the invention allow a complete execution of
the method 100 within the apparatus 10.
[0076] By way of preparation in a step 102 the frame 45 with the
magnetizable pins 40 is moved over the loaded rack 44 for plastic
covers 42 and is lowered, so that one plastic cover 42 is slipped
onto each magnetizable pin 40.
[0077] First the sample to be lysed, contained in a conventional
sampling cavity (not shown in FIGS. 1a and 1b), plus a buffer are
placed 105 in the apparatus 10. The conventional sampling cavity
can e.g. be a tube with blood serum, which in addition bears an
unambiguous identification. This unambiguous identification of the
sample, for example a barcode, rules out any unintentional
confusion of the samples. A method 100, which is to be used in the
diagnosis of biomolecules 28, has to ensure that several samples
can be allocated with absolute certainty and without any danger of
confusion. The apparatus 10 and the method 100 according to the
present invention rule out such confusions of sample material,
since barcode scanners can document the execution of the method 100
in important positions within the apparatus 10.
[0078] The sample is aliquoted 110 into the lysis cavity 22 using a
suitable pipetting device 840, contained in the apparatus 10 (shown
in FIG. 5b). In addition the suitable pipetting device 840 pipets
115 a lysis buffer 30a into the lysis cavity 22.
[0079] All following examples of buffers are used in the
purification procedure for DNA from whole blood; other start
materials partly require other buffer systems. An example for such
a lysis buffer is Lysis Buffer A for blood samples, such as used in
a variety of reaction kits by Invitek. This buffer is based on the
patent DE 19856064C2.
[0080] A lysis procedure 117 takes place at a possibly elevated
temperature provided by the incubation unit 60 accommodating the
lysis cavity 22. A mechanical mixing of the solution 30 in the
lysis cavity 22 during the lysis procedure 117 is possible by
moving the magnetizable pins 40 up and down in the lysis cavity 22.
The lysis procedure 117 takes between 10-30 minutes, depending on
the biomolecules 28. The mixing during the lysis procedure 117 can
selectively take place through cyclical opening of the lid 61 of
the incubation unit 60 or also with an open incubation unit 60.
[0081] During this lysis procedure 117 the apparatus 10 using the
suitable pipetting system 840 prepares cavities 20 arranged in the
accommodation area 81 of the work plate 80 with washing buffers 32a
and 32b and elution buffers 33, preferably first a first washing
buffer 32a, then second a second washing buffer 32b. Without any
limitation more than two washing buffers 32a, 32b are possible.
Furthermore at least one elution buffer 33 is pipetted. Examples
for washing buffers are Wash Buffer I and Wash Buffer II, an
example for elution buffers is Elution Buffer D. These buffers are
based on the patent DE 19856064 C2.
[0082] This is followed by the addition 120 of a binding buffer 30b
and the magnetizable particles 35 to the lysis cavity 22 in
suspension. For example Binding Buffer B6 or Binding Buffer. These
buffers are based on the patent DE 19856064 C2.
[0083] By moving the magnetizable pins 40 of the apparatus 10 up
and down, the biomolecules 28, the binding buffer 30b and the
magnetizable particles 35 are mixed 122 within the lysis cavity 22.
Optionally also the pipetting device 840 itself can be used for
mixing, by absorbing and dispensing the solution. Subsequently the
magnetizable pins 40 are switched to magnetic 125 e.g. by switching
on the current and the magnetizable particles 35 are collected.
[0084] To the magnetizable particles 35 the biomolecules 28 e.g.
nucleic acid 28a have now attached themselves in order to form a
particle-biomolecule complex 36. The magnetizable particles 35 with
the bound nucleic acid 28a are transported 130 by the apparatus 10
through the washing buffers 32a, 32b; according to the following
principle for each washing buffer 32a or 32b etc.
[0085] The magnetizable pins 40 transport 130 the
particle-biomolecule complexes 36 when switched to magnetic and
thereby place the bound nucleic acid 28a in a first cavity 20a or
in a second cavity 20b with the buffer solution 32a, 32b, etc.
Subsequently the magnetizable pins 40 are switched off or the
permanently magnetic pins 40 are removed 135.
[0086] This is optionally followed by a mixing step 140 carried out
once or several times, with switching on the counter magnet 50, and
the particle-biomolecule complexes 36 shift from the switched-off
magnetizable pin 40 to the bottom area 25 of the cavity 20. After
concluding the mixing 145a, 145b of the solution 30 within the
cavities 20a, 20b the counter magnet 50 is switched off again
146.
[0087] Alternatively the particle-molecule complexes 36 are
distributed and mixed 145a with the washing buffer solution 32a
within the first cavity 20a or the particle-biomolecule complexes
36 are mixed 145b with the washing buffer solution 32b within the
second cavity 20b etc. by moving the magnetizable pins 40 up an
down together, of course with the plastic caps or plastic covers 42
disposed on top, so that the pins 40 and the plastic caps 42 dip
into the washing buffer solution 32 within the first cavities 20
once or several times. It would furthermore be possible to carry
out the mixing 145a, 145b of the solution 30 or 32a, 32b etc. by
alternately switching on and off or moving the magnetizable pins 40
and the counter magnets 50. The mixing 145a, 145b of the solution
30 or 32a, 32b in this case was carried out by the
particle-biomolecule complexes 36 within the solution 30. A
movement of the particle-biomolecule complexes 36 between the wall
area or bottom area 25 of the first cavity 20a or the second cavity
20b and the plastic covers 42 would also effect the mixing 145a,
145b of the solution 30. However, this alternative mixing process
is limited by the switching cycles of the counter magnets 50 and
the magnetizable pins 40.
[0088] Switching on 150 the magnetizable pins 40 or inserting the
permanently magnetic pins 40 collects the particle-biomolecule
complexes 36 from the washing buffer 32a, 32b again.
[0089] In an inquiry 160 it is determined whether further washing
buffers 32 etc. are to be processed. In case of further washing
buffers 32 to be processed, this is followed by the transport 130
to the next cavity 20 with the further washing buffer 32.
[0090] However, if according to the inquiry 160 no further washing
buffers 32 have to be processed, the magnetizable pins 40 and the
plastic covers 42 with the particle-biomolecule complexes 36
adhering thereto are lifted from the solution 30 and a waiting step
follows to dry 170 the particle-biomolecule complex 36 at least
partially. Subsequently the dried particle-biomolecule complex 36
is transported to the cavity 20 with the elution buffer 33 and is
placed 180 in the elution buffer 33. Thereupon the magnetizable
pins 40 are switched to non-magnetic or the permanent magnets 40
are removed 190 from the cavities 20.
[0091] This is optionally followed by switching on 200 the counter
magnet 50, whereby the particle-biomolecule complex 36 increasingly
shifts from the switched-off magnetizable pin 40 towards the bottom
area 25 of the cavity 20 due to the counter magnetic field.
[0092] In a subsequent process step a mixing 210 is carried out in
order to dissolve the particle-biomolecule complex 36. For this
purpose the magnetizable pins 40 and the counter magnet 50 are
switched off and the solution 30 with the elution buffer 33 is
mechanically mixed in the cavity 20 within the yield cavity 85 by
moving the pins 40 up and down in a non-magnetic state (with the
magnetic field switched off). Thereby the particle-biomolecule
complex 36 is detached, the particles 36 are brought to suspension
and the biomolecules 28a are desorbed from the magnetizable
particles 35 (this is the so-called elution).
[0093] A subsequent switching on 220 of the magnetizable pins 40
collects the magnetizable particles 35 which are now freed of the
biomolecules 28 from the elution buffer 33. The biomolecules 28 or
nucleic acids 28a remain in the elution buffer 33. In addition a
certain residual amount of the magnetizable particles 35 can remain
in the eluate, which now consists of the elution buffer 33,
biomolecules 28 or nucleic acids 28a and the not completely removed
magnetizable particles 35 which disturb the further processing.
[0094] A disposal 240 of the magnetizable particles 35 removed
upwardly from the elution buffer 33 which still adhere to the
magnetizable pins 40 switched to magnetic takes place e.g. into one
of the cavities 20 used before with the washing buffer 32a, 32b
within the work area 80. The disposal 240 is carried out by
switching off the magnetic field or removing the permanent magnets.
Alternatively the disposal 240 can also take place into the
receptacle 70 (the particles remain on the magnetizable pins 40
first).
[0095] By now switching on 245 the counter magnet 50 of the cavity
20 with the elution buffer 33 the magnetizable particles 35
remaining in the elution buffer 33 are pulled downward and are thus
actively removed from the upper area and are held in the lower area
of the cavity 20. Thereby in the upper area of the cavity 20 with
the eluate 33 an ultraclean eluate is yielded, containing the
biomolecules 28 or the purified nucleic acid 28a.
[0096] By means of the pipetting system 840 this ultraclean eluate
is now removed by pipetting 250 the ultraclean eluate into the
cavities 20 within the yield cavity 85.
[0097] To conclude 270 the method 100 the plastic covers 42 on top
of the magnetizable pins 40 can be slipped off at a rake above the
receptacle 70. If the magnetizable particles 35 remained on the
magnetizable or magnetized pins 40, also the magnetized particles
35 are dropped into the receptacle 70.
[0098] The method 100 can purify all types of biomolecules 28 in
dependence on the magnetizable particles 35, lysis buffer 30a,
binding buffers 30b, washing buffers 32a, 32b and elution buffers
33. With a suitable combination of the washing buffers 32 (32a,
32b, etc.) elution buffer 33 and the individual procedure also
combinations of different biomolecules 28 are possible, for example
DNA and RNA together, or also separately in two cavities or
reaction cavities within the yield cavity 85, or also in two
cavities within two separate yield cavities 85.
[0099] This combination of the pipetting technology with a magnet
separation via magnetizable transport magnets renders possible an
unprecedented repertoire of combined purifications and a greater
variability concerning the sample amounts to be processed.
[0100] The invention contains parallel and serial variants of
upscaling a sample amount. Upscaling means a targeted increase of
the processable sample volume of a start substance. The parallel
and serial variants of the upscaling can also be combined.
Upscaling means that within the framework of the upscaling several
cavities or partly also greater cavities are used. In the several
cavities or the greater cavities the same solution 30 is disposed.
The same solution 30 means that substantially the same sample
material, the same buffer solutions and the same magnetizable
particles 35 are used. In particular the concentrations and/or the
substance amounts of the same solutions 30 are substantially
equal.
[0101] Combining the apparatus 10 and the suitable pipetting system
840 offers the possibility of parallel upscaling. A further aspect
of the apparatus 10 according to the invention is designed for the
parallel processing of 12 samples, wherein in different
configurations also different numbers of samples are possible. The
processing path for the sample or the sample material herein is
referred to as a channel, thus 12 channels are provided. Several of
these purification channels can be used in parallel for one sample,
whereby in this example a sample volume can be processed that is up
to 12 times greater the initial volume. After the preparation of
the eluate with the elution buffer 33 and biomolecules 28 the
pipetting system 840 can control the removal by pipetting 250 in
such a fashion that the eluate from the several channels is placed
in one or several cavities within the yield cavity 85, whereby the
biomolecules 28 distributed to several channels are joined
again.
[0102] Furthermore a serial upscaling can be achieved within the
channels if used for the lysis or incubation 117 within the lysis
cavity 22 an array with several cavities per channel is used.
Therein during the method 100 one channel is used several times
with the same sample, in that the start material is distributed to
several cavities of the lysis cavity 22 within one channel. Then
the particle-biomolecule complexes 36 can be transferred in several
partial steps from these cavities of one channel into the cavity 20
within the work plate 80 into the first washing buffer 32a.
[0103] This means that a serial upscaling within one channel can be
achieved up to a multiple, e.g. the five-fold sample amount. The
objective of this upscaling results from the capacity of the
washing buffers, which can be loaded e.g. with up to the five-fold
amount of the magnetizable particles 35 from one individual
isolation process. Consequently in the case of the combination of
the serial upscaling within the channels and the parallel upscaling
of several parallel channels the sample volume could, in the
extreme case in the example of a 96-cavity microtiter plate, be
increased by 96-fold. This means, starting from the usual amount of
0.2 ml, in the extreme case more than 10 ml could be purified. This
is the amount which is at most contained in e.g. a standard
collection cavity for blood or serum. However, this also means that
per run only one sample can be processed. It is an advantage herein
that no change in equipment is required, it is e.g. possible to use
conventional 96-cavity microtiter plates and uniform plastic covers
42.
[0104] FIG. 3a shows an aspect of a plate with incubator cavities
65. The plate shown in FIG. 3a with the incubator cavities 65 is a
conventional multiwell plate, the cavities of which can accommodate
a volume greater than 2 ml. The incubator cavities 65 of the
multiwell plate can furthermore be arranged in the apparatus 10
along a working direction of the apparatus 10. This working
direction of the apparatus 10 preferably corresponds to the
longitudinal direction (x direction) for moving the frame 45.
[0105] FIG. 3b shows an aspect of a lysis cavity 22 which can also
be used as a work plate 80. Preferably the lysis cavity and/or the
work plate 80 are exchangeable plates, e.g. 96-cavity microtiter
plates or deepwell plates (e.g. disposables). Usually such a
microtiter plate consists of eight rows (A . . . H) of respectively
twelve (1 . . . 12) cavities 65. Of course also different numbers
of the rows and columns or also non-orthogonal arrays of cavities
65 or 20 are possible.
[0106] The serial upscaling within a channel can take place in the
area of the incubation device or the incubation unit 60 within the
lysis cavity 22 in several alternative ways. Selectively a special
plate is used with particularly voluminous (e.g. more than 10 ml),
e.g. elongated incubator cavities 65 (FIG. 3a). In this case the
complete amount of the magnetizable particles 35 is placed into
these incubator cavities 65 and is collected from these cavities
after adsorbing the biomolecules 28 using magnetic force.
[0107] Optionally the transfer of the collected magnetizable
particles 35 out of the lysis cavity 22 into the work plate 80 can
also take place in several sequential steps. Alternatively a lysis
cavity 22 with numerous cavities 20 of a usual size (approx. 1 to 2
ml) can be used e.g. a 96-cavity microtiter plate or a deepwell
plate. Then the sample-reagent mixture to be examined is
distributed to several cavities 65 of this lysis cavity 22 and the
magnetizable particles 35 are first placed into at least one of the
cavities. Subsequently, starting with a first filled cavity, the
magnetizable particles 35 are sequentially transferred to all
cavities filled with this sample-reagent mixture. The magnetizable
particles 35 increasingly take up the biomolecules 28 from all
these filled cavities. At the end of this procedure the magnetic
collection device has collected the biomolecules from all these
filled cavities 65 of the lysis cavity 22 and transferred them to a
first cavity on the work plate 80.
[0108] In a further variant of the serial and parallel upscaling
several parallel channels (e.g. 1 . . . 12) of e.g. several
cavities 65 each (e.g. A-H) of a lysis cavity 22 are filled with
the same sample. The processing steps are then executed one by one,
from incubating to extracting up to finalizing the eluate in one
respectively last cavity on the work plate 80. From the thus
produced last row of cavities with eluate, this eluate can be
transferred from all cavities to at least one cavity on the yield
cavity 85 using the pipetting device 840.
[0109] The invention furthermore provides that within the lysis
cavity for mixing the solutions 30, e.g. in a variant with large
cavities 20 extending in the longitudinal direction of the
apparatus 10, such as the incubator cavities 65 shown in FIG. 3a,
the magnetizable pins 40 are moved not only in the vertical z
direction, but additionally along the longitudinally directed x
direction of the apparatus 10. The magnetizable pins 40 can also be
moved in an oscillating fashion up and down in the vertical z
direction and/or back and forth in the longitudinal direction of
the apparatus.
[0110] FIG. 3c first shows the process steps without the upscaling.
The particle-biomolecule complex 36 is transferred out of the lysis
cavity 22 to individual cavities 20a, 20b of the work plate, as
already described.
[0111] FIG. 3d shows a first variant of the serial upscaling from
an incubator cavity 65 of an incubation plate, as shown in FIG. 3b.
The biomolecule complex 36 is collected from the incubator cavity
65 by moving the plastic covers 42 with the magnetizable pin 40 in
the x direction and z direction and is transported (130) into the
first cavity 20a of the work plate 80.
[0112] FIG. 3e shows a further variant of the upscaling from a
standard plate as lysis cavity 22 into a work plate 80 also with a
standard plate. It is possible to move the magnetizable particles
35 from left to right through the rows A, B, C, . . . H. Upon each
contact of the particles 35 with the solutions in the lysis cavity
22 a yield of biomolecules 28 is increased, which, forming the
particle-biomolecule complex 36, are bound to the magnetizable pins
40 and are thus extracted from the lysis cavity 22. A transfer of
the particle-biomolecule complex 36 in this variant takes place in
one single transport step 130.
[0113] FIG. 3f shows a further variant of the upscaling from the
standard plate as lysis cavity 22 to the work plate 80, which, like
in FIG. 3e, comprises a standard plate. In contrast to FIG. 3e in
FIG. 3f not all rows of the lysis cavity 22 are passed
sequentially. Instead from each of the rows A, B, C, . . . H an
individual transport 130 of the particle-biomolecule complexes 36
takes place into the first cavity 20a of the working plate 80.
[0114] The incubation unit 60 in the alternative apparatus in FIG.
4a to 4c and 5a and 5b is substantially bigger than in FIG. 1a-1c,
so that the plate with the incubator cavities 65 shown in FIG. 3a
can be accommodated by the incubation unit 60. Furthermore the
yield area 86 is no longer arranged on the apparatus 10. The yield
area 86 in this aspect forms part of the overall system 800, as
will be explained below.
[0115] A lid 61 of the incubation unit 60 e.g. is designed in such
a fashion that the lid 61 can be opened automatically, e.g. using a
hinge. In FIG. 4a the lid 61 is shown in a closed state. The lid 61
furthermore optionally has small openings 62 for inserting the
magnetizable pins 40. The frame 45 can be moved across the
incubation unit 60 when the lid 61 is closed and when it is opened,
so that the magnetizable pins 40 have access to the incubator
cavities 65. On the frame 45 a drip-catcher 46 is arranged, which
is best seen in FIGS. 4a and 4c. The drip-catcher 46 moves
underneath the tips 41 of the magnetizable pins 40, whereby a
contamination of cavities 20 by dripping from the magnetizable pins
40 is prevented.
[0116] The first drive unit, a for e.g. comprising a motor 90a and
a band 92a moves the sliding carriage or the frame 45 in the x
direction. The second drive unit b, e.g. comprising of a motor 90b
and a band 92b, opens and closes the lid 61 of the incubation unit
60. A third drive unit c, e.g. with a motor 90c, moves the
arrangement of the magnetizable or permanently magnetic pins 40 and
the plastic caps 42 arranged on top of the pins vertically in the z
direction. A fourth drive unit 90d serves to move the counter
magnets 50.
[0117] For the aspect of the magnetizable pins 40 using electro
magnets the third drive unit 90c for moving the magnetizable pins
40 comprises e.g. a simple motor drive which moves the pins up and
down in the z direction. The magnetic field is then switched on and
off electrically.
[0118] For the aspect of the magnetizable pins 40 using permanently
magnetic arrangements the third drive unit 90c comprises two
motors, wherein at least the magnetically effective portion of the
permanently magnetic arrangement can be removed from the cavities
20 far enough that this equals switching off the magnetic
field.
[0119] FIG. 4d shows a further aspect of the magnetizable pin 40 of
the apparatus 10 in cross section. The magnetizable pin 40
comprises a mantle 40a. On this mantle 40 the plastic cover 42 is
disposed approximately in the lower third. The plastic cover 42
provides the tip 41 of the magnetizable pin 40. By changing the
plastic cover 42 it is possible in a simple fashion to ensure the
cleanliness of the tip 41. The bulge 43 of the magnetizable pins 40
(see FIG. 1a-c) can be omitted in the further aspect of the
magnetizable pin 40.
[0120] The further embodiment of the magnetizable pin 40
furthermore comprises a retaining clip 40g. The retaining clip 40g
serves to hold the plastic cover 44 on the mantle 40a of the
magnetizable pin 40. The retaining clip 40g connects the plastic
cover 44 in a detachable fashion with the mantle 40a of the
magnetizable pin 40. This means that the plastic covers 42 can be
taken up by the mantle 40a and can be shed again reliably.
[0121] As an alternative to the detachable connection of the
plastic cover 42 to the mantle 40a of the magnetizable pin using
the retaining clip 40g the dimensions of the mantle 40a and of the
plastic cover 42 can be adjusted to each other in such a fashion
that a positive fit of the plastic cover 42 on the cap of the
magnetizable pin 40 is achieved.
[0122] On the inside of the mantle 40a of the magnetizable pin 40 a
hollow 40b is disposed. The hollow 40b is suitable to accommodate a
magnetizable element. The hollow 40b and consequently the
magnetizable element can be moved along the longitudinal axis of
the magnetizable pin 40 identified by a dashed line. The mobility
of the magnetizable element serves to control the effect of the
magnetizable element 40b on the tip 41 of the magnetizable pin
40.
[0123] If the magnetizable element 40b is disposed in the area of
the tip 41, the tip 41 is magnetized. This state is also referred
to as "switching on of the magnetizable pin 40". If the
magnetizable element 40b is disposed at a distance from the tip 41,
the tip 41 is not magnetized. This state is also referred to as
"switching off of the magnetizable pin 40". By moving the
magnetizable element 40b between the tip 41 and a position at a
distance from the tip 41a change is possible between switching on
and switching off the magnetizable pin 40. A speed of this change
determines a frequency of the change of a magnetization of the tip
41. The magnetizable element in the hollow 41 can be implemented
either as a permanent magnet or as an electromagnet. Electric
conducts to the magnetizable element in the hollow 41 and a
corresponding voltage supply are required for electromagnets in the
hollow 41.
[0124] It is furthermore conceivable to combine permanent magnets
and electromagnets in the magnetizable pins 40. Thus the
electromagnet could be supplied with current in such a fashion that
the magnetic field of the electromagnet is opposed to and in total
greater than a field of the permanent magnet. Such an aspect would
be of interest if a magnetic pulse is to be used to detach the
biomolecules 28 from the magnetizable pin 40.
[0125] If the magnetizable element 40b is implemented exclusively
as an electromagnet, furthermore the mobility of the magnetizable
element in the hollow 40b relative to the mantle 40a of the
magnetizable pin 40 can be omitted.
[0126] The structure of the magnetizable pin 40 shown in FIG. 4d in
particular allows the movement of the mantle 40a independent of a
movement of the hollow 40b. Consequently, even if a permanent
magnet is used as magnetizable element in the hollow 40b, the
magnetizable pins 40 can be lowered into the cavities 20 in a
switched-off state.
[0127] The apparatus 10 itself has only two axes (x direction
longitudinal and z direction vertical) and does not allow any
translations in the lateral y direction. However, the suitable
pipetting system 840 having at least three axes allows the parallel
and serial upscaling as described above.
[0128] The invention furthermore provides a system 800 for the
so-called "diagnostic purification" of biomolecules 28. The
"diagnostic purification" of biomolecules 28 means that the
regulations and directives of in-vitro diagnostics (IVD) are
fulfilled. The system 800 ensures that no confusion of materials
and substances by a user can occur. Moreover all steps carried out
by the system 800 are comprehensively recorded.
[0129] FIG. 5a shows a top view of the system 800 according to the
present invention. The system 800 comprises the apparatus 10
according to the present invention for purifying biomolecules 28.
This apparatus 10 contains the incubation unit 60 and furthermore
the already discussed elements of the apparatus 10. The elements of
the apparatus 10 are provided with reference numerals in the FIGS.
5a and 5b only if relevant for the description of the system 800.
Concerning all other elements of the apparatus 10 reference is made
to the FIG. 1a-1c and FIG. 4a-4c.
[0130] The system 800 in addition to the apparatus 10 comprises a
loading bay 600 for accommodating substances in the system 800. The
system 800 furthermore comprises a control unit 860 for controlling
the system 800 and the steps carried out by the system 800. The
system 800 furthermore comprises a suitable pipetting system or a
pipetting unit 840. The suitable pipetting system 840 is shown in a
perspective view in FIG. 5b. The pipetting system 840 can be moved
above the system 800 and can sweep the complete base surface of the
system 800. The pipetting system 840 is suitable to transfer the
substances from the loading bay 600 into the apparatus 10 and/or
from the apparatus 10 to the yield area 85. The required pipetting
tips are automatically taken up from the storage cavity 880.
[0131] In the system 800 the yield area 85 was shifted from the
apparatus 10 to an area which is disposed in front of the apparatus
10. This means that the completely purified eluate can be
transferred directly into the yield area or into a yield cavity 85
using the pipetting system 840.
[0132] The loading bay 600 comprises a number of sample receptacles
610 to accommodate sample cavities 620 (best visible in FIG. 7b).
The sample cavities 620 are identified 1031 using the reading
device 500 upon being placed 1030 into the system 800. The reading
device 500 for automatically detecting substances placed into the
loading bay 600 can for example comprise a barcode reader.
[0133] The system 800 comprises a detection device 870 for coded
information about used substances. Substances are the samples,
liquids, solids in their entirety which are used in the course of
the method in the apparatus. The detection comprises the
recognition of at least one coding.
[0134] In the simplest case only the sample coding is recognized in
order to reallocate it in an unaltered and error-free fashion to
the result at the end of the procedure.
[0135] In the alternative case the detection comprises the
recognition of codings of substances, e.g. via barcodes, which can
in particular be components of a so-called kit for purifying
nucleic acids. In this case a check for consistency can be carried
out in order to rule out any confusion within the kit or of the kit
itself.
[0136] As a further alternative also the identification 1031 of the
sample material itself can take place automatically and on the
basis of this coding the selection of the method steps to be
carried out as well as the selection of substances required for
this purpose can be determined via an information system. Through
the reading device 500 the system 800 can in particular identify
1031 every sample cavity 620 and a content of the sample cavity
620.
[0137] An alternative detection variant can also determine the
properties of the used cavities and containers with the cavities
disposed thereon, e.g. via a position of the sample cavity 620 in
the system 800 a cross section of the sample cavity 620 can be
determined. The cross section results from the cross section of the
cylindrical opening in which the sample cavity 620 is inserted. A
height of the sample cavity 620 and consequently a volume of the
sample cavity 620 can be coded on a reading element 501, so that
the volume of the sample cavity 620 becomes part of identifying 510
the sample cavity and the sample content.
[0138] The loading bay 600 furthermore comprises a row of substance
receptacles 650. These consist of cylindrical openings of different
sizes, into which the substance cavities 630 (not shown) can be
inserted. Upon inserting or pushing in 1035 the substance cavities
630 into the loading bay 600 the position of the substance cavity
630 is determined, and thereby its cross section. Furthermore the
content of the substance cavity 620 from the reading element 501 is
identified 1036 by the reading device 500. The height of the
substance cavity 620 can also be coded on the reading element 501.
This is of interest, in particular in the case that substance
cavities 620 of different heights can be placed 630 in the loading
bay 600.
[0139] In the case that very large amounts of samples have to be
purified, it is conceivable that the sample material is placed 1035
into the system 800 in one of the substance receptacles 650. The
reading device 500 will correctly identify 1031 the inserted sample
amount as sample material.
[0140] The system 800 furthermore optionally comprises a plurality
of sensors 830 to record and log a plurality of parameters 835.
Such a parameter can be the temperature of the incubation unit 60
and the dwell time of the sample material in the incubation unit
60, but is not limited thereto. Furthermore a plurality of
parameter controls 839 is provided to control the plurality of
parameter values in the system 800.
[0141] The system 800 is for e.g. suitable to determine required
binding buffers 30b, washing buffers 32 and elution buffers 33 as
well as the required magnetizable particles 35 on the basis of the
identified sample material via the identification of the
substances. Furthermore the system 800 is adapted to determine a
required substance amount for each of the elements of the group.
Through the identification of the substances it is on the other
hand also possible, without interpreting the coding of the sample,
to determine the process parameters on the basis of the substances
used in the apparatus and to control the actions of the apparatus
and the method steps accordingly.
[0142] The system 800 can furthermore, on the basis of the
identified sample material and/or on the basis of the used reagents
and aids, determine a particular IVD-conforming sequence of steps
and further process parameters for the diagnostic purification of
the biomolecules 28 and for e.g. also the required documentation.
Likewise on the basis of the detected information the number of
steps required for the diagnostic purification and the selection of
further reagents and/or aids can be determined (e.g. the type of
magnetizable particles 35).
[0143] FIG. 5b shows a perspective view of the system 800 according
to the invention. In this representation the sample cavities 620
are recognizable particularly well.
[0144] FIG. 6a shows an extension of the method 100 carried out as
a method 900 for the diagnostic purification of biomolecules 28.
For the method 900 the method 100 of FIG. 2 is extended. More
specifically, FIG. 8a shows an extension of the step 102 for taking
up the plastic covers 42 on the magnetizable pins 40 in the method
100. If the method 100 is to be extended in the form of the method
900, step 102 has to be expanded.
[0145] In a step 1005 first a plate with incubator cavities 65 is
loaded into the incubation unit 60. Subsequently in a step 1010
first a work plate 80 is loaded into the work-plate accommodation
81 of the apparatus 10.
[0146] This is followed by an enquiry 1012 to check whether plastic
covers 42 have to be refilled in the racks 44. In the case that a
refill is necessary, the racks 44 are refilled in a step 1015.
[0147] Subsequently in a step 1020 the work plate 80 is loaded with
the required elution solutions and/or the washing buffers. Finally
it is checked in an inquiry 1021 whether the receptacle 70 for the
used plastic covers 42 has to be emptied. If required, the emptying
is carried out with step 1025.
[0148] In a step 1030 the sample cavities are placed into the
loading bay 600. Therein the identification of the inserted sample
cavities takes place in step 1031. Subsequently in a step 1035 the
substance cavities are placed into the loading bay 600, whereupon
the inserted substance cavities are identified in step 1036.
[0149] On the basis of the identified sample material and/or the
identified substances the system 800 determines in step 1040 a
suitable multiwell plate and loads this into the incubation unit
60. Therein a required incubation volume is taken into account.
[0150] This is followed by step 1045, where the information read by
the reading device 500 is recorded and transmitted to the control
unit 860 and/or the documentation unit 850. Furthermore step 1045
allows a consistency check of the identified substances in the
loading bay 600. Thereby for e.g. the use of a false buffer
solution with a sample material can be prevented, so that falsely
positive or falsely negative results of the purification can be
prevented.
[0151] This is followed by a step 1050. In the step 1050 required
process parameters 850 are determined.
[0152] FIG. 6b shows the individual stages of the step 1050. First
in a step 1055 the required substances are determined among the
identified substances in the loading bay 600. Subsequently in a
step 1060 a respective substance amount is determined for each of
the required substances to carry out the method 900. The substances
involved in the method 900 are at least one element of the
substances discussed above.
[0153] In a step 1065 the required parameter values 835 are
determined on the basis of the identified substances. This is
followed by step 1070 in which the required process steps are
determined on the basis of the identified substances.
[0154] Subsequently in step 1075 the apparatus 10 is controlled by
the control unit 860 of the system 800. The control unit 860
monitors the apparatus 10 during the execution of the method 100.
Step 1075 also comprises the monitoring of the parameter values 835
during the execution of the method 100.
[0155] If the method 900 is carried out, the step 105 (FIG. 2) for
supplying the sample can be omitted, since the sample material was
already supplied in step 1030 (FIG. 6a).
[0156] FIG. 7 shows a block diagram of the system 800 according to
the invention. The system 800 comprises a parameter control 839 to
control a plurality of parameter values 835. The parameter values
835 are detected by a plurality of sensors 830. Furthermore the
system 800 comprises the apparatus 10 according to the invention, a
pipetting device 840 as well as a documentation unit 850 for
documenting detected information, for example by the reading device
500. The information read by the reading device 500 is recorded in
a recording module 870 and forwarded to the system control 860
and/or the documentation unit 820. The control unit 860 controls
and regulates the system 800 on the basis of programs and the
recognized information about the substances. The elements of the
system 800 shown in FIG. 7 are connected to each other and
communicate with each other.
[0157] The system 800 comprises software for controlling the method
shown in FIGS. 8a and b. This software is run by a microprocessor
and can be programmed in any suitable programming language.
* * * * *